I. N. Rocas, J. F. Siqueira Jr, A. F. B. Andrade &M. Uzeda
Department of Endodontics, Faculty of Dentistry, Estacio de Sa University
Department of Microbiology, State University of Rio de Janeiro, and Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil.

Introduction.
The association of spirochaetes with oral diseases has been known for many years. Amongst the oral spirochaetes, nine species have been cultured and named. Four species have been cultured widely and reliably by many laboratories: T. denticola, T. pectinovorum, T. socranskii andT. vincentii (Chan & McLaughlin 2000). Recently, 16S rRNA-based analyses have allowed the description and characterization of additional cultivable oral treponemes: T. malthophilum (Wyss et al. 1996), T. medium (Umemoto et al. 1997), T. amylovorum (Wyss et al. 1997), T. lecithinolyticum (Wyss et al. 1999) and T. parvum (Wyss et al. 2001).
Oral treponemes, particularly the species T. denticola, T. socranskii, T. pectinovorum and T. vincentii, have been reported to be associatedwithgingivitis, adult periodontitis, acute necrotizing ulcerative gingivitis, localized juvenile periodontitis, refractory periodontitis and HIV associated periodontal diseases (Socransky & Haffajee 1997, Moter et al. 1998,Willis et al. 1999, Dewhirst et al. 2000). It has been revealed that the periodontal pocket harbours a highly diverse treponeme population (Choi et al.1994, Dewhirst et al. 2000).Whilst treponemes are usually detected in periodontally diseased sites, some species are only infrequently found at periodontally healthy sites (Moter et al.1998,Willis et al.1999,Dewhirst et al.2000).This indicates a possible causal relationship.
Miller (1894) was the first to report the occurrence of spirochaetes in endodontic infections. Brown &Rudolph (1957) observed spirochaetes in 14% of their pulp samples using dark field and phase-contrast microscopy. Also using darkfield microscopy, Thilo et al. (1986) reported 6% of spirochaetes amongst the root canal microbiota. With the use of light and transmission electron microscopy, Nair (1987) revealed that spirochaetes seemed to form a significant component of the root canal microbiota. In a dark field and scanning electron microscopic study, Dahle et al. (1993) described a spirochaete isolated from a root canal that was much longer (140 mm in length) and thicker (2 mm in diameter) than usual for oral treponemes. van Winkelhoff et al. (1985) reported the presence of spirochaetes in pus samples from acute periradicular abscesses. Darkfield examination of the exudate from periodontal and endodontic abscesses found that in periodontal abscesses the spirochaete count ranged from 30 to 60%, whereas in endodontic abscesses the range was 0-10%(Trope et al.1984).I n situ scanning electron microscopic investigations have also disclosed spirochaetes in infected root canals (Molven et al.1991, Siqueira et al. 2002).
Although spirochaetes had already been observed in infected root canals by microscopy, they have only recently been identified. Dahle et al. (1996) reported that four spirochaete strains isolated from endodontic infections were found to have similarities to T. pectinovorum, but probably represented two previously unidentified newspecies. Smallwood et al. (1998) reported the occurrence of spirochaetes in all samples collected from infected canals using PCR, but did not characterize which species of spirochaetes were involved. Recently, the occurrence of T. denticola in cases of endodontic infections by means of molecular methods has been reported (Siqueira et al. 2000a,b, 2001a, Rocas et al. 2001). The checkerboard DNA-DNA hybridization method has been used to investigate the prevalences of 42 bacterial species in infections of endodontic origin, and T. denticola was found in17.9% of the cases (Siqueira et al.2000b). When the more sensitive single PCR method was used, this species was found in 34.5% of the canals associated with asymptomatic chronic periradicular lesions, 53.3% of the cases of acute apical periodontitis and in 50%of the teethwithacute periradicular abscess. In general, T. denticola was found in 42.6% of the cases (Siqueira et al.2001a).Roc¸as et al. (2001) found relatively high prevalence of T. denticola in endodontic infections, sometimes forming a complex with Bacteroides forsythus and Porphyromonas gingivalis. Taken together, these reports suggested that T. denticola can participate in the pathogenesis of periradicular diseases. Dot-blot hybridization with species-specific oligonucleotide probes and amplified bacterial DNA has recently allowed the detection of T. maltophilum and T. socranskii in root canal infections (Jung et al. 2001). T. socranskii was also recently detected in pus samples from acute periradicular abscesses by checkerboard DNA-DNA hybridization (Siqueira et al. 2001b).
Studies have revealed that PCR methods are more sensitive, rapid and accurate than the culture method for the detection of anaerobic bacteria (including spirochaetes) in saliva and subgingival plaque samples (Ashimoto et al. 1996, Sakamoto et al. 2001). Therefore, the purpose of this study was to investigate the prevalence of four Treponema species, T. denticola, T. socranskii, T. vincentii and T. pectinovorum, in root canal infections using a nested PCR assay.

Materials and methods.

Specimen sampling.
Thirty-two single-rooted teeth from adult patients, all of them having carious lesions, necrotic pulps and radiographic evidence of periradicular disease, were included in this study. Of these, 22 cases had chronic asymptomatic periradicular lesions and 10 cases were diagnosed as acute apical periodontitis (six cases showing both spontaneous symptoms and tenderness to percussion and four cases showing only tenderness to percussion). The teeth had no periodontal pockets over 4 mm deep. The ages of the patients ranged from 18 to 60 years.
Samples were collected using strict asepsis. The tooth was cleansed with pumice and isolated with a rubber dam. The tooth and the surrounding field were then cleansed with 3% hydrogen peroxide and decontaminated with a 2.5% sodium hypochlorite solution. Access cavities were prepared using sterile burs without water spray. The operative field, including the pulp chamber, was then swabbed with 2.5% sodium hypochlorite. This solution was inactivated with sterile 5%sodiumthiosulphate. If the root canal was dry, a small amount of sterile saline solution was introduced into the canal. Samples were initially collected by means of a size 15 K-type file (Dentsply Maillefer, Ballaigues, Switzerland) with the handle cut off. The file was introduced to a level, approximately1mmshort of the tooth apex, based on diagnostic radiographs and a discrete filing motion was applied. Afterwards, two sequential paper points were placed to the same level and used to soak up the fluid in the canal. Each paper point was retained in position for 1min. The cut file and the two paper points were then transferred to cryotubes containing 1mL of 5% dimethyl sulphoxide in trypticase-soy broth (Difco, Detroit, MI, USA) (TSB-DMSO). Samples were immediately frozen at _20 8C.

DNA extraction.
The samples in TSB-DMSO were thawed to 37 8C for 10 min and vortexed for 30 s. Microbial suspensions were washed three times with100 mL of double-distilled water by centrifugation for 2 min at 2500 g. Pellets were then resuspended in 100 mL of double-distilled water, boiled for10 min and chilled on ice. After centrifugation to remove cell debris for10 s at 9000 g at 4 8C, the supernatant was collected and used as the template for PCR amplification.
Reference DNA from T. denticola B1 (Forsyth Dental Center),T. pectinovorum ATCC 33768,T. socranskii S1 (Forsyth Dental Center) and T. vincentii ATCC 35580was also extracted to serve as positive control for the primers used.

Oligonucleotide primers.
Primer sequences are shown in Table 1. Universal primers anneal at conserved regions near the 50- and 30- ends of 16S rDNA, generating a practically full-length 16S product. Sequences for T. denticola and T. socranskii consisted of specific forward and reverse primers as described by Ashimoto et al. (1996) and Willis et al. (1999), respectively. Primers for T. pectinovorum and T. vincentii utilized an universal forward sequence (positions 8-27 of E. coli16S rDNA) with a species-specific reverse sequence as described by Willis et al. (1999). Primers were purchased from Oligos Etc. Inc. (Wilsonville, OR, USA).

Table 1. PCR primer pairs used for detection of four treponemal species in infected root canals.

Nested PCR.
Five microlitres of the supernatant from clinical samples was used as target in the PCR reaction using universal 16S rDNA primers. One microlitre of the universal reaction was then used as template for each nested specific reaction. PCR amplification was performed in 25 mL of reaction mixture containing 0.2 mm concentration of forward and reverse primers, 2.5 mL of10X PCR buffer, 1.5 mm MgCl2, 1.25 U of Taq DNA polymerase (Gibco BRL, Gaithersburg, Md, USA) and 25 mm concentrations of each deoxyribonucleoside triphosphate (dATP, dCTP, dGTP and dTTP) (Gibco BRL). PCR reactions were performed in 25-well microtitre plates.
Preparations were amplified in a DNA thermocycler (Primus 25/96, MWG-Biotech, Ebersberg, Germany). The PCR temperature profile included an initial denaturation step at 97 8C for1min, followed by 26 cycles of a denaturation step at 97 8C for45 s, a primer annealing step at 55 8C (for the universal16S rDNA primer),60 8C (for T. denticola primer), 53 8C (for T. pectinovorum or T. socranskii) or 56 8C (for T. vincentii) for 45 s, an extension step at 72 8C for 1min and a final step of 72 8C for 4 min.
Eight microlitres of each PCR product were electrophoresed on a 1.5% agarose gel at 4 Vcm_1 in Tris- borate-EDTA buffer. The gel was stained for 15 min with 0.5 mg mL_1ethidiumbromide and photographed under ultraviolet light. Positive reactions were determined by the presence of bands of the appropriate sizes (Table 1). Both 100 bp and 1 kb DNA ladder digests (Gibco BRL) were used to size markers.

After PCR amplification using universal primers for the 16S rDNA, a product of the appropriate size (1505 bp) was obtained from all samples examined, indicating that bacteria were present in all cases and demonstrating the suitability of the DNA for PCR and the absence of inhibitors in the reaction mixture.
In the 22 cases of asymptomatic periradicular lesions, T. denticola was detected in17 teeth (77.3%), T. socranskii in nine (40.9%), T. vincentii in four (18.2%) and T. pectinovorum in three (13.6%). Nineteen cases (86.4%) yielded at least one of the four species. In the cases of acute apical periodontitis showing spontaneous pain, T. denticola was found in five of six cases (83.3%) and T. socranskii in three of six (50%). None of these cases was positive for either T. vincentii or T. pectinovorum. In the total cases diagnosed as acute apical periodontitis,T. denticola was detected in eight cases (80%), whilst T. socranskii and T. vincentii were detected in four (40%) and one case (10%), respectively. No symptomatic case yielded T. pectinovorum. Eight cases were positive for at least one of the test species.
In general, species-specific nested PCR detected T. denticola in 25 of 32 cases (78.1%), T. socranskii in 13of 32 cases (40.6%),T. vincentii in five of 32 cases (15.6%) and T. pectinovorum in three of 32 cases (9.4%). At least one of the four Treponema species was found in 27 of the 32 cases examined (84.4%). Data regarding prevalence values are represented in Fig.1. The species T. denticola was associated with T. socranskii in 12 cases (eight asymptomatic and four symptomatic cases), with T. vincentii in four cases (three asymptomatic and one symptomatic cases) and with T. pectinovorum in two asymptomatic cases. T. socranskii was detected together with T. pectinovorum in two asymptomatic cases and with T. vincentii in two cases (one asymptomatic and another symptomatic).No case yielded a coinfection with T. pectinovorum and T. vincentii. Three cases yielded three of the treponemal species evaluated herein. The species T. denticola, T. socranskii and T. vincentii were concomitantly infecting two root canals (one asymptomatic and another symptomatic). Another asymptomatic case was positive for T. denticola, T. socranskii and T. pectinovorum.

Figure 1. Prevalence of four Treponema species in root canal infections. Data refer to the percentage of asymptomatic cases, symptomatic cases and the total number of cases positive for each target species. Data about Treponema refer to the cases where at least one of the four species was detected.

Discussion.
Data on microbial morphology provides few clues for the identification of most microorganisms, and physiological traits are often ambiguous (Relman 1999, Chan & McLaughlin 2000). In addition, several microorganisms are difficult or even impossible to grow under laboratory conditions (Relman 1999). These factors are especially true in the case of spirochaetes (Dewhirst et al. 2000). The nested PCR method used in this study was a 16S rDNA-based assay in which practically the entire 16S rDNA was amplified in the first reaction using universal primers followed by a second round of amplification using species-specific nested primers, internal to the first PCR product, which were used to check for the presence of the target species. The advantages of using16S rDNA analysis is that this molecule is found in all bacteria, it appears to lack artifacts of lateral transfer between contemporaneous microorganisms, is large enough to provide a significant number of nucleotides to compare sequences and yet it is small enough to conveniently analyse (Woese1987).The16S rRNA of bacteria has been extensively examined and sequenced and has been used to determine phylogenetic relationships amongst living organisms. As a consequence, data from 16S rDNA sequences have been widely used for accurate and rapid identification of known and even unknown bacterial species, using techniques that do not require microbial cultivation. This makes it possible to rationally design primers covering taxon-specific signatures, which permits microbial identification with high sensitivity and specificity, without the need to cultivate the microorganism. The rRNA gene-based PCR has proved to be superior to culture in clinical situations, such as infections caused by microorganisms with unusual growth requirements that are difficult or even impossible to culture and specimens taken during antimicrobial treatment (Relman 1999, Dewhirst et al. 2000, McPherson & Moller 2000, Sakamoto et al. 2001). PCR methodology is at least10-100timesmoresensitive than the other sensitive microbiological identification methods (Zambon & Haraszthy1995). Nested PCR has increased sensitivity and possibly also increased specificity when compared with single PCR (McPherson & Moller 2000). The assay used in this study had the detection limit of10-100 cells of the target species in the sample. A 5% volume of the original sample (100 mL) was used in individual PCR experiments. Considering sample dilution, the detection limit was approximately 200-2000 cells in the whole sample, which is still more sensitive than practically all other identification methods with regard to fastidious anaerobic bacteria. There is no agreed bacterial load for the induction of a periradicular lesion, and the method of assay should therefore detect the smallest number of possible bacterial cells in root canal samples. Thus, one of the greatest advantages of the PCR methodology as used in the present study.
In the present study, T. denticola was clearly the most prevalent amongst the four species tested. Whereas this bacterial species has been detected in 17.9 and 42.6% of the infected root canals by means of checkerboard DNA-DNA hybridization (Siqueira et al.2000b) and single PCR assay (Siqueira et al. 2001a), respectively, the nested PCR used in this study allowed the detection of T. denticola in 78.1% of the cases examined. Such discrepant findings can be explained by the different detection limits of the methods used. The nested PCR used in the present study is more sensitive than the single PCR assay previously used, which is more sensitive than the checkerboard DNA-DNA hybridization.
Jung et al. (2001) evaluated the occurrence of species of oral treponemes in 79 teeth associated with periradicular lesions by using PCR amplification with bacterial universal primers and subsequent dot-blot hybridization. They found T. maltophilum in 26% and T. socranskii in 2.7%of the cases. No case yielded the species T. denticola, T. pectinovorum and T. vincentii. These findings are diametrally divergent from those reported in this study. Differences between the results could have occurred for a variety of reasons. For instance, the nested PCR used in this study is more sensitive than the dot-blot hybridization used by Jung et al. (2001). In addition, the possibility exists that differences may have also been due to geographical differences in the composition of the oral microbiota and consequently of the root canal microbiota.
Data from the present study revealed that at least one of the four target species was present in 86.4% of the asymptomatic cases and in 80% of the cases diagnosed as acute apical periodontitis. In general, treponemes were found in 84.4% of the cases examined and the possibility exists that this figure could be increased if other species had been also tested. With regard to treponemes, these are the highest figures hitherto reported by identification studies for root canal infections. In addition, it appears that no previous studies had yet detected T. vincentii, a potential periodontal pathogen, in infected root canals.
The four Treponema species evaluated in the present study have been implicated in a variety of oral diseases and have been demonstrated to be pathogenic microorganisms. Kesavalu et al. (1997) investigated the virulence characteristics of T. denticola, T. pectinovorum, T. socranskii and T. vincentii following subcutaneous injection in mice. All species induced well-demarcated, dose-dependent, raised subcutaneous abscesses, which were similar in time of onset, lesion progression and duration of healing. Alterations in the environment may influence the pathogenicity of oral treponemes (Kesavalu et al. 1999). It has been demonstrated that different species of oral treponemes can elicit proinflammatory cytokine production by host cells and this stimulation does not necessarily require live microorganisms. In a study where human gingival fibroblast were challenged by Treponema species, Nixon et al. (2000) observed that dead cells of T. pectinovorum generally induced a twofold greater level of IL-6 and IL-8 than the live bacteria. Several virulence factors have been described and suggested for the four species evaluated, most particularly for T. denticola (the best characterized and more easily cultivated oral spirochaete). They include factors involved in adherence to host cells and tissues, tissue invasion ability, direct and indirect tissue damage and evasion of host defense mechanisms (Sela et al. 1987, Grenier 1991, Klitorinos et al.1993, Umemoto & Namikawa1994, Haapasalo et al. 1996, Scott et al.1996, Fenno & McBride 1998, Yang et al. 1998, Peters et al.1999, Rosen et al.1999,Nixon et al.2000, Grenier & Mayrand 2001, Heuner et al. 2001).

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